Tritiated alumina, prepared by exchange of the surface hydroxyl groups with tritiated water, is a suitable reagent for exchange-labeling of specific compounds in low concentrations prior to chromatographic analysis. In a chromatographic column, it detects and measures submicrogram quantities of material

Erenrich, E. H.

Klein, P. D.

English

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September 1967	 Brief 67-10315 
AEC-NASA TECH BRIEF 
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of the U.S. AEC or from AEC-NASA interagency efforts. They are issued to encourage commercial 
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the Clearinghouse for Federal Scientific and Technical Information, Springfield, Virginia 22151. 
Tritiated Alumina Serves as Reagent for Self-Labeling Analysis 
The problem: 
To develop a method of exchange-labeling of 
specific compounds in low concentrations prior to 
chromatographic analysis. The requirements for the 
achievement of such an analytical method are: (I) the 
preparation of a uniform, reproducible absorbent of 
known specific activity and exchange capability; (2) 
the establishment of the linearity of exchange reaction 
with the sample size; (3) knowledge of the degree to 
which all exchangeable positions in the molecule 
participate in the labeling process; and (4) the effective 
resolution of one class member from another by the 
chromatographic column. 
The solution: 
Tritiated alumina, prepared by exchange of the 
surface hydroxyl groups with tritiated water, is a 
suitable reagent. When put in a chromatographic 
column, it effects the labeling of compounds passed 
over it. Columns of this material provide the ability 
to detect and measure submicrogram quantities of 
material. The use of radioactive hydrogen permits the 
gleaning of information through radioactive meas-
urements that previously required mass spectrometer 
measurements. 
How it's done: 
One hundred grams of alumina were heated at I-mm 
pressure for 2 hours, cooled in vacuo, and deactivated 
with 3 ml of tritiated water. The rehydrated alumina 
was permitted to equilibrate and again was subjected 
to heat at reduced pressure. This time, and all subse-
quent times, the water obtained during the dehydra-
tion process was collected; the volume and specific 
activity of this collected water were measured. Usually 
four to six cycles were sufficient to achieve complete 
equilibration of the alumina surface with the tritiated 
water used in rehydration.
The specific activity of the water removed by high 
temperature dehydration is only an approximate 
indication of the specific activity of the alumina 
surface, particularly when the number of cycles is 
small. Therefore, a small portion of the alumina 
prepared in each cycle was set aside to be used for a 
test chromatographic column. Coprostanone was used 
as the test sample, and the total activity per mg was 
measured by liquid scintillation counting after com-
plete elution from the column. This procedure pro-
vides a means of standardizing the specific activity by 
comparison to a known reference. 
In this process, the previously unlabeled constitu-
ents of a suitable class became radioactive during their 
passage through, and separation by, the chromato-
graphic column to a degree that reflects both their 
concentration and those molecular features susceptible 
to labeling. To determine whether a proportionate 
response could be obtained over a wide range of 
sample sizes, the reference sample (a ketosteroid) was 
run at levels from 10 mg to 3kg, and the recovery of 
activity was determined. The results indicated that a 
direct linear relationship exists between the weight of 
sample applied and the amount of radioactivity ap-
pearing in the chromatographic peak. The results also 
indicated that the tritiated alumina may be diluted with 
unlabeled alumina of the same adsorbent activity to 
obtain sample labeling activities that are directly 
proportional to the dilution. 
There are two alternative capabilities of self-label-
ing analysis: (I) knowing the constituents of a mix-
ture, their individual concentrations can be de-
termined radiochemically; or (2) knowing the amounts 
of each, the degree of substitution on the alpha 
carbons can be ascertained by determining the speci-
fic activity as a multiple of the adsorbent activity. 
(continued overleaf) 
-I
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Notes: 
1. A mixture of ketosteroids was tested using 30% 
benzene/pentane. The area under each peak was 
totaled and the recovery of the samples calculated 
from the relationship: Sample weight = (c.p.m. in 
peak) x (molecular weight/(aliquot) x (efficiency) 
x (surface specific activity) x (theoretical number 
of enolic H) x (2.22 x 10). The results showed 
good recovery. 
2. In addition to ketosteroids, this method is applica-
ble to other detonic compounds which can be 
chromatographic on alumina. 
3. It is not necessary to know the specific activity of 
the HTO used in the preparation of the reagent 
or the number of times the absorbent has been 
equilibrated. 
4. The detection limits of columns of tritiated alumina 
appear to extend to submicrogram quantities. 
5. Additional details are contained in Analytical 
Chemistry, vol. 38, March 1966, p. 480-484.
6. Inquiries concerning this innovation may be di-
rected to:
Office of Industrial Cooperation 
Argonne National Laboratory 
9700 South Cass Avenue 
Argonne, Illinois 60439 
Reference: B67-10315 
Source: P. D. Klein and E. H. Erenrich
Biological and Medical 
Research Division
(ARG-209) 
Patent status: 
Inquiries about obtaining rights for commercial use 
of this innovation may be made to: 
Mr. George H. Lee, Chief 
Chicago Patent Group 
U.S. Atomic Energy Commission 
Chicago Operations Office• 
9800 South Cass Avenue 
Argonne, Illinois 60439 
Brief 67-10315	 Category 03


Tritiated alumina serves as reagent for self-labeling analysis

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